Human metapneumovirus (HMPV) was first reported in 2001 and has quickly come to be recognized as a significant agent of respiratory tract disease worldwide, especially in the pediatric population, in immunocompromised individuals, and in the frail elderly. We are using recombinant DNA methods to characterize viral molecular biology and pathogenesis and to develop attenuated derivatives of HMPV for use as a live intranasal pediatric vaccine.[unreadable] HMPV is an enveloped virus with a genome that is a single negative-sense strand of RNA of approximately 13.3 kb. We previously developed the first complete HMPV consensus sequence and characterized the two genetic subgroups, A and B. Molecular studies showed that HMPV encodes eight mRNAs that are translated into nine proteins (the M2 mRNA contains two separate overlapping open reading frames encoding two distinct proteins, M2-1 and M2-2). By analogy to human respiratory syncytial virus, its better-known relative, the HMPV proteins are: N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion protein; M2-1, RNA synthesis factor; M2-2, RNA synthesis factor; SH, small hydrophobic protein; G, attachment glycoprotein; and L, viral polymerase.[unreadable] We developed a reverse genetic system for HMPV whereby complete infectious virus can be generated in cell culture entirely from cloned cDNAs, whereby the genome can be engineered for basic studies and designing vaccines. [unreadable] We found that four viral genes could be deleted individually and in various combinations with little or no effect on viral replication in vitro, namely: G, SH, M2-1 and M2-2. The del-SH, del-G and del-SH/G viruses were described in the previous year. The del-M2-1 virus did not replicate detectably in hamsters, indicating that it likely is over-attenuated for vaccine purposes. The del-M2-2 virus was 4000-fold reduced in replication in African green monkeys compared to wild type, making it an attractive vaccine candidate. Each of these deletion mutants also has been made in a version that also expresses GFP; these are being used to evaluate infection and host response in an in vitro model of differentiated epithelium and in monocytes, macrophages and dendritic cells.[unreadable] The del-M2-2 virus exhibited an increase in the accumulation of mRNA normalized to the genome template, suggesting that M2-2 ordinarily has a role in the negative regulation of transcription. Unexpectedly, investigation of induction of type I interferon by HMPV provided evidence that the M2-2 protein is an antagonist of the interferon system. Specifically, expression of M2-2 results in an inhibition of the activation of Interferon Regulatory Factor 3, but did not appear to affect activation of NK-kB.[unreadable] A change in the viral mRNA gel pattern also was observed for the M2-1 virus. Specifically, the mRNA species visualized on a Northern blot appeared to migrate as less-diffuse, more-compact bands. Further evaluation, including studies with a mini-replicon system, showed that the mRNAs made in the absence of M2-1 contained polyA tails that were approximately 25 nucleotides in length compared to the 150-250-nucleotide length that is characteristic of eukaryotic mRNAs. Thus, HMPV M2-1 appears to have a role in the synthesis or stability of the polyA tail. [unreadable] Northern blot analysis showed that the various HMPV genes differ considerably with regard their representation in monocistronc mRNA versus readthough mRNAs. Studies with a mini-replicon system showed that differences in the efficiency of termination appeared to be due completely to differences in the gene-end signal. The gene-end signals of the M2 and SH genes are particularly inefficient, resulting in very low levels of expression of the downstream SH and G genes. Alignment of the various gene-end signals indicated that this was due to only one or two nucleotide differences in the signal. [unreadable] We evaluated the biological significance of these inefficient signals by making recombinant viruses in which an efficient signal (from the M gene) and an inefficient signal (SH) were swapped in various combinations. These viruses have been recovered and are presently being analyzed in vitro and in hamsters. The results to date indicate that any change from the natural arrangement was associated with a decrease in plaque size. [unreadable] Chimeric versions of recombinant HMPV were generated by replacing the N or P open reading frame with its counterpart from the closely related avian metapneumovirus (AMPV) subgroup C. Surprisingly, in Vero cells, wild-type AMPV replicated to an approximately 100-fold higher titer than HMPV. Also surprisingly, the N and P chimeric viruses replicated to a peak titer that was 11- and 25-fold higher than that of parental HMPV. The basis for this effect is not known but was not due to obvious changes in the efficiency of gene expression. In hamsters, the N and P chimeras were approximately 100-fold attenuated compared to HMPV but nonetheless induced a high level of neutralizing serum antibodies and protective efficacy against HMPV. In African green monkeys (a better model for the natural human host), the mean peak titer of the P chimera was reduced 100- and 1000-fold in the upper and lower respiratory tracts, whereas the N chimera was reduced only 10-fold. Both chimeras were comparable to wild type HMPV in immunogenicity and protective efficacy. [unreadable] Thus, we have generated to date four viruses that are promising vaccine candidates, namely the del-G, del-SH/G, del-M2-2, and HMPV/AMPV-P viruses. The latter two viruses, together with wt HMPV, have been produced under conditions appropriate for products for human use and will be amplified to make clinical trial material for phase I studies of safety, attenuation and immunogenicity in humans. The P chimera is an attractive candidate because it combines improved growth in vitro with attenuation in vivo. The del-M2 virus is attractive because ablation of an interferon antagonist might increase the immunogenicity of the virus. [unreadable] We investigated the contribution of cleavage-activation of the F protein of HMPV to replication and pathogenicity in rodents and nonhuman primates. Recombinant HMPVs were generated in which the naturally-occurring trypsin-dependent cleavage sequence (R-Q-S-R/) was replaced by sequences whose cleavage in vitro does not depend upon added trypsin. Two of these were multi-basic sequences derived from AMPV type A (R-R-R-R) or type C (R-K-A-R), with the former containing the consensus furin protease cleavage motif (R-X-R/K-R/). The third one (R-Q-P-R) was derived from a recently described trypsin-independent HMPV isolate. For safety reasons, the modifications were done in an HMPV variant that was attenuated by the deletion of SH and G. Each of the introduced cleavage sequences conferred trypsin-independent cleavage and growth to HMPV in vitro. However, they differed in the efficiency of growth and plaque formation in vitro in the absence of trypsin, in the following order of decreasing efficiency: R-R-R-R , R-K-A-R, R-Q-P-R. During multi-cycle growth, replication of the R-R-R-R mutant was not augmented by added trypsin, indicative of highly efficient trypsin-independent cleavage. When inoculated intranasally into hamsters, there was essentially no difference in the magnitude of replication in the upper or lower respiratory tract, and no virus was detected in organs outside of the respiratory tract. Evaluation of the most cleavage-efficient mutant, R-R-R-R, in African green monkeys showed that there was no detectable change in the magnitude of replication in the upper and lower respiratory tract or in immunogenicity and protective efficacy against HMPV challenge. These results suggest that cleavage activation is not a major determinant of HMPV virulence. The use of a trypsin-independent backbone in vaccine manufacture will be a significant improvement.